Chloroform induction of ornithine decarboxylase activity in rats.

Chloroform is a drinking water contaminant that has been demonstrated to be carcinogenic to mice and rats resulting in an increased incidence of liver and kidney tumors, respectively. The mechanism of chloroform carcinogenicity might be by tumor initiation and/or promotion. Since induction of ornithine decarboxylase (ODC) activity has been proposed as a molecular marker for tumor promoters, we have investigated the effect of chloroform on ODC activity in rats. Chloroform induced a dose-dependent increase of hepatic ODC with an apparent threshold at 100 mg/kg body weight. Female rats were two to four times more susceptible to to chloroform. Upon daily dosing of chloroform for 7 days the liver became less susceptible, with the last dose of chloroform resulting in only 10% of the activity observed after a single dose. Nuclear RNA polymerase I activity was also induced by chloroform. Chloroform, rather than increasing the activity of renal ODC, resulted in a 35% reduction. The induction by chloroform of hepatic ODC activity might be associated with regenerative hyperplasia while the renal carcinogenicity of chloroform could not be demonstrated to be associated with ODC induction.


Introduction
Chloroform has been demonstrated in mice and rats to increase the incidence of hepatocellular carcinoma and epithelial tumors of the kidney, respectively (1). The initiation of carcinogenesis is believed to involve a somatic mutation; however, chloroform was nonmutagenic in the Ames Salmonella/microsome assay (2), in E. coli K 12 for basepair substitution (3), and in the 8-azaguanine locus in Chinese hamster lung fibroblast in cell culture (4). It also appeared that the amount of covalent binding of chloroform to rat liver and kidney DNA was very minimal (5). The negative evidence for chloroform mutagenicity and the low level of DNA interaction has resulted in the proposal that chloroform is not an initiator of carcinogenesis but is rather a tumor promoter.
Chloroform has long been known to be toxic in the liver and kidney, which vi'e the organs in which an increased incidence of tumor formation has been found (1,6,7). The hepatotoxicity include fatty degen-*Health Effects Research Laboratory, U.S. Environmental Protection Agency, Cincinnati, Ohio 45268. eration, glycogen depletion, vacuolation, swelling and necrosis. The severe tissue damage that resulted in regenerative growth of the liver (8) could stimulate the progression of preneoplastic cells to tumors. This proposed epigenetic stimulation of the neoplastic progression is called promotion.
Ornithine decarboxylase (ODC) induction has been associated with tumor promotion in skin (9) and liver (10), so that it might be a molecular marker for tumor promotion. ODC is the first and rate-limiting enzyme in polyamine biosynthesis (11) and has a very rapid turnover time of 10-20 min in liver (12,13). Drugs, hormones, and partial hepatoectomy which increased cellular proliferation have been shown to increase ODC activity (14). In this communication, we report the effect of chloroform on ODC activity in rat liver and kidney.

Subcellular Fractionation
Animals were sacrificed by decapitation. The livers or kidneys were removed, rinsed, blotted, weighed and homogenized in 1.0 volume (1 ml/g tissue) of ODC homogenizing buffer (0. 1M NaH2PO4, 0.8mM pyridoxal-5-phosphate, 2.0mM EDTA; pH 7.5) at 0°C with a glass homogenizer fitted with a Teflon pestle. The homogenate was centrifuged at 10,000g for 10 min at 4°C. The supernatant fraction was decanted off and further centrifuged at 144,000g for 60 min at 4°C. The 144,000g supernatant fraction was removed with a Pasteur pipet introduced below the surface to avoid the turbid lipid layer on top. This cytosol fraction was used as the enzyme source for the ornithine decarboxylase assay.
Liver nuclei used in the RNA polymerase I assay were prepared by the isolation procedure developed by Muramatsu et al. (15) and described by Yu and Feigelson (16).

Enzyme Assays
All animals for the ornithine decarboxylase assay were sacrificed between 9 and 10 AM. Cytoplasmic ODC activity was measured by determination of the release of 14CO2 from ornithine (1-14C) essentially as described by Bethell and Pegg (17). The cytoplasmic fraction (100 [lI) was incubated with 900 ,ul of ODC incubation medium containing 56mM NaH2PO4, 3.8mM dithiothreitol, 2.3mM EDTA (pH 6.5) and 1.0 ,uCi of DL-ornithine (1-14C) (51.3 mCi/ mmole). After incubation for 30 min at 37°C the reaction was stopped by the addition of 0.3 ml of 5M H2SO4, and the 4C02 released was trapped on filter paper discs saturated with 0.25 ml of hyamine hydroxide. The production of 14CO2 was linear with respect to the amount of enzyme protein added to each incubation. The discs were removed, placed in liquid scintillation vials containing 15.0 ml ACS (Amersham) and 0.41 ml of 0.5N HCl, and the radioactivity determined in a Beckman LS 8100 liquid scintillation counter (Beckman Instrument Inc., Palo Alto, Calif.). The background activity, determined with acid denatured cytosol, was subtracted in each case. Protein was measured by use of the Bio-Rad protein assay kit (Bio Rad, Rich-SAVAGE ET AL. mond, Calif.). Enzyme activity was expressed as CO2 evolved/mg protein/30 min incubation.
RNA polymerase I activity was determined by using purified intact nuclei as the enzyme source as described by Roeder and Rutter (18). Isolated nuclei were incubated in a final volume of 1.0 ml containing 10 mg pyruvate kinase (400 units/mg protein), 23 ,umole Tris-HCI (pH 7.9), 1.0 ,umole MnCl2, 4.0 ,umole KCI, 3.0 ,umole NaF, 2.0 ,umole phosphoenol pyruvate, 0.8 ,umole 2-mercaptoethanol. 0.3 ,umole each GTP, CTP, ATP, 0.06 ,umole unlabeled UTP, 0.002 ,umole 3H-UTP (20.3 Ci/,umole), and 50 ,umole ammonium sulfate and with and without lmM ot-amanitin (19). Following incubation for 10 min at 30°C, the reaction was terminated by the addition of 4.0 ml of 10% TCA. The samples were centrifuged at 3,000 rpm for 10 min in a Beckman TJ-6 table top centrifuge (Beckman Instrument Inc., Palo Alto). The supernatant was discarded and the pellet washed two times with 3.0 ml of 5% TCA. The pellet was resuspended in 3.0 ml of 5% TCA and collected under vacuum onto HA 0.45mM Millipore filters. The filters were washed with 10 ml of 5% TCA, followed by 5 ml of 60% ethanol and allowed to air-dry overnight. The next day they were oxidized in a Packard Model 306 sample oxidizer (Packard Instrument Co., Downers Grove, Ill.). Radioactivity was determined by liquid scintillation counting and protein measured as described above. The data were expressed as pica moles of 3H-UTP incorporated/10 min/mg protein.

Results
The effect of various carcinogens and tumor promoters on ornithine decarboxylase (ODC) activity in male rat liver was determined (Table 1). With the exception of benzene and saccharin, all of the compounds tested stimulated ODC. Chlorinated carcinogens and tumor promoters such as chloroform, carbon tetrachloride and the polychlorinated biphenyl, Aroclor-1254, caused the greatest increase in ODC activity.

Chloroform Time Course and Dose Response
The time course of chloroform induction of ODC in male rat liver was determined (Fig. 1). Enhancement was evident as early as 2 hr and slowly increased until 18 hr, at which time maximum enhancement of ODC activity occurred. The maximum enhancement of ODC activity was 43.5-fold control level and 74 pmole C02/30 min/mg protein. By 24 hr, the peak enhancement of ODC activity had decreased by 80%. cEnhancement of ODC activity compared to the mean of eight saline-treated rats which was 1.7 ± 0.44. dStatistically greater than the activity in saline-treated rats by the Student t-test at the level of p < 0.01.
The administration of a daily dose of chloroform for 7 days resulted in a lower enhancement of ODC activity compared to a single dose (Fig. 2). After seven total doses of chloroform, the ODC activity was only 10% the activity observed after the first dose. The decreased responsiveness of the liver to chloroform was even apparent for a second dose of chloroform administered on the following day.
The dose-response relationship of ODC induction by chloroform in female and male rats was performed (Fig. 3). Female rats were two to four times as sensitive to chloroform as male rats. There was an apparent threshold in male rats for ODC induction below 100 mg/kg body weight. The ODC activity increased with dose of chloroform until 750 mg/kg body weight in both female and male rats. Higher doses of chloroform could not be tested in females due to a high incidence of lethality.

Renal ODC Activity
The ODC activity of male rat kidney was approximately 300-fold the activity in male rat liver ( Table  2). The function of this high level of ODC activity in male kidney is unknown; however, after unilateral nephrectomy, a fourfold increase of ODC activity occurred 24 (20). Chloroform (750 mg/kg body weight), which resulted in a 43.5-fold enhancement of rat liver ODC activity (Table 1), caused a 35% reduction of kidney ODC activity (Table 2).
Hormonal Modulation of Chloroform-Induced ODC Adrenalectomy prevented the increase in rat liver ODC activity that resulted from treatment with barbiturates ( Table 3). The extent of chloroform induction of ODC activity was not statistically diminished by prior adrenalectomy. Therefore, unlike barbiturates, the enhancement of rat liver ODC by chloroform was not dependent on the adrenal gland. Pretreatment with propranolol (a beta adrenergic receptor blocker) or 17-ot-hydroxyprogesterone (a corticosterone receptor blocker) also did not diminish the increased level of ODC induced by chloroform.

RNA Polymerase I Response to Chloroform
RNA polymerase I is another enzyme that has been demonstrated to increase during the hyperplastic response in rat liver (14). Chloroform induction of RNA polymerase I activity was determined 'The dose of propranolol was 10 mg/kg body weight and of chlorofrom 750 mg/kg body weight.
gStatistically decreased from intact animal by the Student t-test at the level of significance of p < 0.01. in the presence of lmM a-amanitin ( Table 4). The a-amanitin was used to inhibit RNA polymerase II and III (Table 4). Chloroform compared to saline treated rats resulted in a 2.3-fold enhancement of liver nuclear RNA polymerase I activity.

Discussion
The induction of hepatic ODC has been demonstrated to be associated with the hyperplastic and regenerative response to drugs, hormones and partial hepatectomy (21). Chloroform has been demonstrated to induce a regenerative hyperplasia in rat liver (8). Chloroform caused a dose-dependent enhancement of hepatic ODC that could be associated with the regenerative hyperplasia. An apparent threshold for chloroform induction of ODC activity was indicated below 100 mg/kg body weight which might indicate a threshold for regenerative hyperplasia. Female rats compared to males exhibited a 2-to 4-fold greater response of ODC induction. Chloroform also induced hepatic nuclear RNA polymerase I, another enzyme that has been associated with the hyperplastic response in liver (14). The induction of ODC and hyperplasia has been proposed as molecular markers for tumor promoters (9).
Tumor promotion appears to require the prolonged and repeated administration of the promoter (9). Upon daily administration of chloroform, the extent of the enhancement decreased so that after a total of seven doses, the ODC activity was only 10% the level after a single dose. This reduction in the effect of chloroform could have resulted from the inhibition of accumulated putrescine (22). The hyperplastic response of a-hexachlorobenzene in the liver as measured by the increase in DNA replication, was resistant to a second dose (23). The hyperplastic response of chloroform as indicated by the reduced enhancement of ODC might also be limited.
The kidney was the target organ of chloroform carcinogenicity in rats. Kidney ODC activity was not induced by chloroform but was rather reduced. The level of ODC activity in kidneys was approximately 300-fold the hepatic level. Even in presence of this high control level, unilateral nephrectomy was capable of inducing ODC in the regenerating kidney (24). Therefore, the carcinogenicity and possible tumor promoting activity of chloroform in rat kidney would appear not to be associated with the induction of ODC. In summary, the hepatic regenerative hyperplasia induced by chloroform in rats appeared to be associated with the induction of ODC while the renal carcinogenicity was not associated.